Methods for determinging the influence of protein binding on antiretroviral activity

ABSTRACT

The present invention relates to methods for determining the influence of human plasma or serum protein binding on antiretroviral activity at physiologically achieved conditions, by using specific virus strains in a cell-based antiviral assay.

This application claims priority benefit of U.S. Provisional ApplicationNo. 60/358307 Feb. 22, 2002, the contents of which are expresslyincorporated by reference herein.

BACKGROUND OF THE INVENTION

Following administration, drugs are transported in biological fluids(e.g. in blood) partly in solution as free drug and partly bound toblood components (e.g., plasma or serum proteins, blood cells). Thephysiologically active substances are in equilibrium between a free formand a form bound to endogenous ligands present in the same fluids (seereviews by Kremer, et al. Pharmacol Rev. 1988, 40:147). Only free drugis available for passive diffusion to target tissue sites where thedesired biological activity may take place. When compared to thetotal-substance level, the free drug concentration is more closelyrelated to drug concentration at the active site, to drug effects, andto clinical effectiveness. As such it is generally considered that theunbound fraction of drug is pharmacologically active (Levy, G.: Effectof plasma or serum protein binding of drugs on duration and intensity ofpharmacological activity. J. Pharm. Sci. 65, 1264-1265 (1976)) and thatthe bound fraction is not immediately available for distribution throughthe body or for certain routes of elimination.

Theoretically, there is a direct proportional relationship between thetotal plasma concentration and the unbound fraction as long as thesaturation level of the finite number of binding sites on the plasmaproteins is not reached. For most drugs given within the normal clinicaltherapeutical ranges, the protein concentration to which a drug may bindis much higher and therefore, saturation of potential binding sites ishardly ever exceeded or even approached.

However, in practice, there are many exceptions to this rule, such as,for instance, salicylate, disopyramide, phenylbutazone, naproxen,valproic acid, whose total plasma concentrations exhibit an inverselylinear relationship with the binding to plasma or serum proteins, i.e.the higher the total plasma concentration, the lower the bound fraction.

Thus, binding to plasma or serum proteins may have significant changesin the unbound fraction of certain drugs, which may further translate ineffects on the distribution, pharmacological activity and rate ofelimination of the same.

Along this line, large changes in kinetic parameters associated tochanges in plasma or serum protein binding are frequently cited topredict some important alteration in clinical effect. SeePharmacokinetic and Pharmacodynamic Data Analysis, Concepts &Applications, J. Gabrielsson and D. Weiner, 3^(rd) Ed, SwedishPharmaceutical Press. However, when changes in binding are associatedwith clinical effects, it has almost always been found that this is theresult of a change in unbound drug clearance caused by a mechanism quiteindependent of plasma or serum protein binding (Holford NHG. Clin.Pharmacokinetic. 29: ppl 139 (1995)). Winter et al. (Basic ClinicalPharmacokinetics, 3^(rd) ed., Applied Therapeutics, (1994)) concludedthat there is little evidence demonstrating that monitoring unbound druglevels improves the correlation between the plasma or serumconcentration and the pharmacologic effect or therapeutic outcome. Seealso Levy G. (In Drug-Protein binding. Edited by Reidenberg MM, ErrillS. Praeger, New York, 1986).

Nevertheless, the question whether plasma or serum protein binding hadan effect on in vivo activity of anti-HIV compounds has recentlyheightened a great interest in the investigations of effects of serumproteins on activity and pharmacokinetics of antiviral compounds invitro (Billello et al. 1995, J. Infect. Dis. 171:546-551; Bilello et al.1996, Antimicrobiol. Agents Chemother. 40:1491-1497; Lazdins et al.1996, J. Infect. Dis. 175:1063-1070; Kiriyama et al. 1996, Biopharmac.Drug Dispos. 17:739-751; Zhang et al. 1999, J. Infect. Dis.180:1833-1837; Jones et al. 2001, Br J Clin Pharmacol. 51:99-102;Kageyama et al. 1994, Antimicrob Agents Chemother. 22:499-506), and invivo (Sadler et al. 2001, Antimicrob. Agents Chemother. 45:852-856).

In general, it has been asserted that high levels of protein binding ofantivirals lead to poor clinical efficacy. As illustration, the study invitro by Bilello et al. (1995, J. Infect. Dis. 171:546-551; 1996,Antimicrobiol. Agents Chemother. 40:1491-1497) demonstrated that theantiviral efficacy of two HIV protease inhibitors (PIs), A77003 andA80978, decreased as the concentration of α₁-acid glycoprotein (AAG) wasincreased and that the inhibition of HIV protease was highly correlatedwith the amount of intracellular inhibitor. Similarly, the clinicalsignificance of these effects in vitro was shown by the lack of clinicalefficacy of the HIV PI SC-52151, which has potent antiretroviralactivity in vitro but insufficient activity in vivo, because extensiveprotein binding prevented intracellular diffusion (Fischl et al. JAcquir Immune Defic Syndr Hum Retrovirol 1997, 15:28-34).

Despite of a considerable literature regarding the effect ofprotein-binding on antiretrovirals, most methods for calculating theeffect of plasma or serum protein binding, are focused on quantifyingthe unbound fraction of drug by employing common physico-chemicalprotocols, including equilibrium dialysis, ultrafiltration,ultracentrifugation, and gel filtration; and indirect methods such asdetermination of drug in saliva, or measurement of blood/plasma or serumor erytlhrocyte/plasma or serum drug concentration ratios. Thus,existing methods do not allow to measure the plasma protein binding ofdrugs with physiological conditions.

Although it has been argued that the free drug concentration, or unbounddrug levels, is more closely related to drug concentration at the activesite, to drug effects, and to clinical effectiveness, the monitoring ofunbound drug levels still fails to find a relationship between plasma orserum concentration and the pharmacologic effect or therapeutic outcome,see Winter and Levy et al. above.

In view of the clinical significance and the medical need topharmacokinetically characterize HIV inhibitors, a convenient andreliable method to measure the functional effect of protein binding onthe efficacy of drugs is the subject of this invention. Accordingly, thepresent invention provides a method for measuring the influence ofplasma or serum protein binding on antiretroviral therapy by use of acell-based antiviral assay. This method has proved successful in findinga relationship between drug plasma or serum concentrations in thepresence of plasma or serum proteins, with pharmacologic effects. Thiscorrelation is possible when using specific viral strains as means fortesting at drug concentrations that fall within the range of drug plasmaconcentrations present at physiological conditions, i.e. in patients. Byusing such viral strains, one is able to test at a physiological and ata measurable range of effective concentrations, that is, in thephysiological zone where a dose-response curve may be obtained.

The method of the present invention is additionally advantageous as itis a reliable simplification of the in vivo environment of an antiviralagent, as well as an approximate reproduction of the complexity of humanplasma or serum It further takes into consideration the in vivoequilibrium and/or ready-state kinetics experienced by the antiviraldrugs with the viruses. It additionally ponders the different mechanismsand stages of binding kinetics exhibited by the drugs with theirligands, i.e. the accumulation kinetics of concentration-dependentbinding to tissues, the linear (constant free fraction) orconcentration-dependent (increasing free fraction with increasing drugconcentration) binding to plasma or serum proteins.

The present invention provides a method for pharmacokineticallycharacterizig HIV inhibitors in the presence of plasma or serumproteins, that is by determining therapeutic amounts and subsequentdosage regimens, resulting in more accurate and effective therapeuticamounts and dosage regimens, ultimately translating in an improvedtreatment for HIV infected patients.

The method subject of this invention allows as well for an improvedpreclinical evaluation and selection of new antivirals for futureclinical development.

Furthermore, often there may be competition between drugs in plasma orserum protein binding, in which agents that are bound tightly, such ascoumarin anticoagulants, macrolide or lincosamide antibiotics that bindtightly to α₁-acid glycoprotein (AAG), are able to displace less tightlybound compounds from their binding sites and thus can increase the freeform of the drug and improve the biological efficacy (Sommadossi, etal., 1998 U.S. Pat. No. 5,750,493). Therefore, the present inventionprovides as well a method for selecting compounds that competitivelybind with plasma or serum proteins, said selection being useful forco-administering agents to compete for plasma or serum protein binding,so that an increase of the free plasma or serum concentration ofantiretrovirals is achieved. Alternatively, highly potent HIVinhibitors, eventually with a narrow therapeutic range, may furtherbenefit from the co-administration of compounds that enhance binding ofHIV inhibitors with plasma or serum proteins, so that their workingconcentrations are maintained free from toxic levels, thus preventingovermedication. Consequently, the present invention includes as well amethod for selecting compounds that enhance binding of antiretroviralswith plasma or serum proteins.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the influenceof human plasma or serum protein binding on antiretroviral activity atphysiologically achieved conditions, by making use of specific virusstrains in a cell-based antiviral assay.

The present invention provides as well a method for pharmacolineticallycharacterizing HIV inhibitors in the presence of plasma or serumproteins, that is by determining therapeutic amounts and subsequentdosage regimens, for various purposes including, lead optimisation, drugselection, preclinical evaluation, and clinical optimisation.

The present invention further concerns a method for selecting compoundsthat competitively bind with plasma or serum proteins as well as amethod for selecting compounds that enhance binding of antiretroviralswith plasma or serum proteins, said selections being useful forco-administering agents to compete for or enhance plasma or serumprotein binding, aiming to an improved management of therapeuticconcentrations of HIV inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the influence of AAG on the anti-HIV activityof saquinavir

FIG. 2 is a graph showing the influence of AAG on the anti-HIV activityof indinavir

DETAILED DESCRIPTION

The present invention relates to a method for determining the influenceof human plasma or serum protein binding on antiretroviral activity.More specifically, the present invention provides a method forestablishing the phenotypic variability of antiretroviral therapy in thepresence of human plasma or serum proteins at physiologically achievedconditions, that is by maklng use of specific virus strains.

Thus, the present invention provides a method for determining theinfluence of human plasma or serum protein binding on antiretroviraltherapy, comprising:

-   -   i) determining the inhibitory activity of at least one HIV        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one HIV        inhibitor in a cellular assay in the absence of the human plasma        or serum proteins against the at least one HIV virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and ii); and iv) determining the influence of human plasma        or serum protein binding on said at least one HIV inhibitor        based on the ratio obtained in iii); and    -   wherein said at least one HIV virus strain has been selected to        be inhibited by said at least one HIV inhibitor with an        inhibitory activity which falls within the range of plasma        concentrations of said at least one HIV inhibitor when used at        therapeutic dosages.

In a similar embodiment, the present invention provides a method fordetermining the influence of human plasma or serum protein binding onantiretroviral therapy, comprising:

-   -   i) determining the inhibitory activity of at least one protease        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one        protease inhibitor in a cellular assay in the absence of the        human plasma or serum proteins against the at least one HIV        virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and id); and    -   iv) determining the influence of human plasma or serum protein        binding on said at least one protease inhibitor based on the        ratio obtained in iii); and    -   wherein said at least one HIV virus strain has been selected to        be inhibited by said at least one protease inhibitor with an        inhibitory activity which falls within the range of plasma        concentrations of said at least one protease inhibitor when used        at therapeutic dosages.

Any cell based assay capable of measuring changes in the ability of apathogen or malignant cell to grow in the presence of a therapeuticagent(s) can be used in the present invention. Such assays ofphenotyping include all methods known to persons of skill in the art. Asan illustrative example, methods for phenotyping viruses suitable foruse in the present invention include, but are not limited to, plaquereduction assays, PBMC p24 growth inhibition assays (see, e. g., Japouret al., Antimcrob. Agents Chemother. 37: 1095-1101 (1993); Kusumi etal., J. Virol. 66: 875-885 (1992), both of which are expresslyincorporated herein by reference), recombinant virus assays (see, e. g.,Kellam & Larder, Antimicrob. Agents Chemother. 38: 23-30 (1994);Hertogs, et al. Antimicrobial Agents and Chemotherapy (1998), 42(2),269-276; and Pauwels et al., 2nd International Workshop on HIV DrugResistance and Treatment Strategies, Laake Maggiore, Italy. Abstr. 51(1998), all of which are expressly incorporated herein by reference);the use of GFP as a marker to assess the susceptibility of anti-viralinhibitors (Marschall et al., Institute of Clin. and Mol. Virol.,University of Erlanger Nuremberg, Schlobgarten, Germany); and cellculture assays (Hayden et al., N. Eng. J. Med. 321: 1696-702 (1989),herein incorporated by reference).

As yet other illustrative examples, cellular assays for phenotypingmalignant cells suitable for use in the present invention include, butare not limited to, flow cytometric assays (see, e. g., Pallis et al.,Br. J. Haematol., 104 (2): 307-12 (1999); Huet et al., Cytometry 34 (6):248-56 (1998), both of which are expressly incorporated herein byreference), fluorescence microscopy (see, e. g., Nelson et al., CancerChemother. Pharmacol. 42 (4): 292-9 (1998), expressly incorporatedherein by reference), calcein accumulation method (see, e. g., Homolyaet al., Br. J., Cancer. 73 (7): 849-55 (1996), expressly hereinincorporated by reference), and ATP luminescence assay (see, e. g.,Andreotti et al., Cancer Res. 55 (22): 5276-82 (1995), expresslyincorporated herein by reference).

The measurement of the influence of plasma or serum protein binding onthe efficacy, of various antiretrovirals may be used in concert withdirect cell based phenotyping assays, for example, Antivirogram™ (Virco,Inc.; WO 97/27480, U.S. Pat. No. 6,221,578 incorporated herein byreference). Combined with HIV virus strains of various degrees ofresistance or sensitivity, the present method allows the measurement ofantiviral inhibitory activities in the presence and absence of plasma orserum proteins and determination of the efficacy of said antiretroviralsat physiological conditions.

Accordingly, the phenotypic drug sensitivity, or phenotypic drugresistance of the HIV virus strains to one or more HIV inhibitor(s) inthe presence of plasma or serum proteins at physiological conditions isexpressed as antiviral inhibitory activity, or effective concentrations.This is then compared to the inhibitory activity for the same assaycomponents but in the absence of plasma or serum proteins. Thephenotypic drug sensitivity or resistance of the sampled virus strain toeach therapy in the presence of plasma or serum proteins is thenexpressed in terms of a fold-change in inhibitory activities, e.g. IC₅₀values, IC₉₀ values, EC₅₀ values, EC90 values, etc., compared to theinhibitory activities obtained with the absence of said plasma or serumproteins.

“Susceptibility” or “sensitivity” to a therapy refers to the capacity ofthe disease, malignant cell, and/or pathogen to be affected by thetherapy. “Resistance” refers to the degree to which the disease,malignant cell, and/or pathogen is unaffected by the therapy. Thesensitivity, susceptibility or resistance of a disease towards a therapymay be expressed by means of an inhibitory activity or effectiveconcentration value. The inhibitory activity is the concentration atwhich a given therapy results in a reduction of the pathogen's growthcompared to the growth of the pathogen in the absence of a therapy. Theeffective concentration is the concentration of an inhibitor thatproduces the maximal possible effect As such, the EC₅₀ or EC₉₀ value isthe effective drug concentration at which 50% or 90% respectively of theviral population is inhibited from replicating. The IC₅₀ or lC₉₀ valueis the drug concentration at which 50% or 90% respectively of the enzymeactivity is inhubited. Accordingly, other fractions are possible andrange up to 100% such as, 90%, 75%, 50%, 30%, etc. Here in thisinvention, both terms will be referred as inhibitory activity.

Efficacy, also known as intrinsic activity, is used to describe themaximum effect of a drug.

In order to be able to test the influence of plasma or serum proteinbinding on the anti-HIV potency of compounds at physiologically achievedconcentrations, thus at those concentrations of drug actually present inthe patients, one needs to employ HIV viral strains of various degreesof resistance or sensitivity. Thus, viral strains for which theinhibitory activity of said anti-HIV compounds, when determined in theabsence of human plasma or serum proteins, falls within the range ofplasma drug concentrations of said compounds in patients. Accordingly,inhibitory activity values corresponding to clinically relevantconcentrations, sub-therapeutic concentrations and in vitro testingconcentrations are determined for the different viral strains, and thosevirus strains exhibiting clinically relevant inhibitory activityconcentrations will be selected and employed in the methods of thisinvention. Preferably a maximum or worst-case measurable resistanceshould be obtained where a given inhibitor still has an effect. Thisconcentration is dependent on the intrinsic activity of the drug and onthe level of resistance of the virus strain.

Resistance of a disease to a therapy may be caused by alterations inphenotype or genotype. The resistant ‘behaviour’ of the virus is acombined result of the effects of many different mutations and thecomplex interactions between them, including genetic changes that havenot even been identified yet. Genotypic alterations include mutations,single nucleotide polymorphisms, microsatellite variations, epigeneticvariations such as methylation.

The influence of human plasma or serum protein binding on antiretroviralactivity is defined as the change in inhibitory activities ratio, e.g.IC₅₀, IC₉₀, EC₅₀, EC₉₀ values ratios, the inhibitory activities beingdetermined in the presence and in the absence of plasma or serumproteins. The influence of human plasma or serum protein binding on acertain drug is a measure of the variation of the potency exhibited bythe drug.

Potency is a measure of the relative sensitivity on the concentration(or dose) axis in producing a particular effect.

Physiological conditions means the same range of therapeutic plasmaconcentrations as found in the human body. Physiological conditions alsoinclude those blood components at those concentrations that are found inthe human body. For instance, physiological conditions comprises theaddition of human serum at 50% concentration, alpha₁-acid glycoproteinin a range from 0.5 to 2 mg/ml, or human serum albumin at around 45mg/ml.

Physiological plasma drug concentrations may be obtained frombibliographical data The different virus strains and level of resistanceor sensitivity that they exhibit, in terms of increase of inhibitoryactivities, EC₅₀ values, or EC₉₀ values may also be found in theliterature, or by running antiviral experiments. As an example, proteaseinhibitors have clinically relevant concentrations in a range of around500 nM and more, sub-therapeutic concentrations in the range of around50 nM to around 500 nM, and in vitro testing concentrations in the rangeof less than around or 50 nM. Viral strains, for which HIV inhibitorsexhibit EC₅₀ values of interest for the methods of the present inventionare for example R13020, T13127, T13025, LAI, for which saquinavirexhibits EC₅₀ values of around 7 μM, 710 nM, 166 nM, and 15-4.7 nM,respectively. For viral strains R13020, R13025, R13031, and LAI,ritonavir exhibits EC₅₀ values of around 27 μM, 2 μM, 407 nM, and 36 nM,respectively. For viral strains R13025, R13028, and LAI, indinavirexhibits EC₅₀ values of around 2 μM, 261 nM, and 36 nM, respectively.For viral strains R13127, R13036, R13028, and LAI, nelfinavir exhibitsEC₅₀ values of around 8 μM, 1 μM, 254 nM, and 32 nM, respectively. Forviral strains R13127, R13025, and LAI, amprenavir exhibits EC₅₀ valuesof around 2 μM, 412 nM, and 42 nM, respectively. For viral strainsR13273, R13485, and LAI, lopinavir exhibits EC₅₀ values of around 2 μM,367 nM, and 9 nM, respectively.

The term binding refers to an interaction or association between aminimum of two entities, or molecular structures, such as a ligand andan antiligand. The interaction may occur when the two molecularstructures are in direct or indirect physical contact or when the twostructures are physically separated but electromagnetically coupledthere between, e.g. by hydrogen bonds or Van der Waals interactions.Examples of binding events of interest in a medical context include, butare not limited to, ligand/receptor, antigen/antibody, enzyme/substrate,enzyme/inhibitor, protein/protein, DNA/DNA, DNA/RNA, RNA/RNA, nucleicacid mismatches, complementary nucleic acids, nucleic acid/proteins,plasma or serum proteins/drugs, nucleic acids/drugs.

HIV inhibitors comprise nucleoside reverse transcriptase inhibitors(NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), proteaseinhibitors (PIs), entry inhibitors, fusion inhibitors, gp41 inhibitors,gp120 inhibitors, integrase inhibitors, co-receptors inhibitors (e.g.CCRS, CXCR4, . . . ), budding/maturation inhibitors, etc.

HIV nucleoside reverse transcriptase inhibitors include those compoundswhose mechanism of action comprises an inhibition of the viral reversetranscriptase enzyme. As example, and with no limitation to existing andfuture new compounds, HIV nucleoside reverse transcriptase inhibitorsinclude zidovudine (AZT), lamivudine (3TC), stavudine (d4T), zalcitabine(ddC), didanosine (ddI), abacavir (ABC)

HIV non-nucleoside reverse transcriptase inhibitors include thosecompounds whose mechanism of action comprises an inhibition of the viralreverse transcriptase enzyme. As example, and with no limitation toexisting and future new compounds, HIV non-nucleoside reversetranscriptase inhibitors include nevirapine, delavirdine, efavirenz,TMC120, TMC125, capravirine, calanolide, UC781, SJ-1366, benzophenones,PETT compounds, TSAO compounds.

HIV nucleotide reverse transcriptase inhibitors include those compoundswhose mechanism of action comprises an inhibition of the viral reversetranscriptase enzyme. As example, and with no limitation to existing andfuture new compounds, HIV non-nucleotide reverse transcriptaseinhibitors include adefovir (PMEA), tenofovir (PMPA), and otherphosphonates.

HIV protease inhibitors include those compounds whose mechanism ofaction comprises an inhibition of the viral protease enzyme. As example,and with no limitation to existing and future new compounds, HIVprotease inhibitors include ritonavir (RTV), indinavir (IDV), nelfinavir(NFV), amprenavir (APV), telinavir (SC-52151), tipranavir (TPV),saquinavir (SQV), lopinavir (LPV), atazanavir, palinavir, mozenavir, BMS186316, DPC 681, DPC 684, AG1776, GS3333, KNI-413, KNI-272, L754394,L756425, LG-71350, PD161374, PD173606, PD177298, PD178390, PD178392, PNU140135, maslinic acid, U-140690, R0033-4649, TMC114, TMC126, theirprodrugs, metabolites, N-oxides and salts.

HIV entry inhibitors or gp120 inhibitors include those compounds whosemechanism of action comprises an inhibition of the viral entry or gp120glycoprotein. As example, and with no limitation to existing and futurenew compounds, HIV entry or gp120 inhibitors include BMS806, dextransulfate, suramin, chicoric acid.

HIV fusion inhibitors or gp41 inhibitors include those compounds whosemechanism of action comprises an inhibition of the viral fusion or gp41glycoprotein. As example, and with no limitation to existing and futurenew compounds, HIV fusion or gp41 inhibitors include T20, T1249.

HIV integrase inhibitors include those compounds whose mechanism ofaction comprises an inhibition of the viral integrase enzyme. Asexample, and with no limitation to existing and future new compounds,HIV integrase inhibitors include L-870810, L-870812,pyranodipyrimydines, S-1360.

Co-receptors inhibitors include those compounds whose mechanism ofaction comprises an inhibition of the interaction of HIV with cellularreceptors present on the cell membrane (e.g. CCR5, CXCR4). As example,and with no limitation to existing and future new compounds,co-receptors inhibitors include TAK 779, AMD3100, AMD8664, AMD070,SHC-C, SHC-D, AK602, TAK-220, UK-427,857, T22.

HIV budding/maturation inhibitors include those compounds whosemechanism of action comprises an inhibition of the viralbudding/maturation. As example, and with no limitation to existing andfuture new compounds, HIV budding/maturation inhibitors include PA-457.

By HIV herein is generally meant HIV-1. However, the invention is alsoapplicable to HIV-2.

Plasma or serum proteins include all proteins found endogenously inplasma or serum. Examples of plasma or serum proteins include withoutlimitation Albumin (HSA), Alpha-1-acid Glycoprotein (AAG),Alpha-1-Antichymotrypsin, Alpha-1 Antitrypsin AT, alpha-fetoprotein,Alpha-1-microglobulin A1M, Alpha-2-Macroglobulin A2M, Angiostatin,Beta-2-Glycoprotein 1, Beta-2-microglobulin, Beta-2-Microglobulin B2M,Beta-N-Acetylglucosaminidase B-NAG, recombinant Centromere Protein B,Collagens (type 1-VI), Complement Clq, Complement C3, Complement C4,Ceruplasmin, Chorionic Gonadotrophin HCG, Chorionic Gonadotrophin BetaCORE BchCG, C-Reactive Protein CRP, CK-MB (Creatine Kinase-MB), CK-MM &CK-BB, Cystatin C, D-Dimer, dsDNA, Ferritins, Glycogen Phosphorylase ISOBB, Haptoglobulin, IgA, IgE, IgG, IgG, IgM, Kappa light chain, lambdalight chain, recombinant LKM Antigen, La/SS-B, Lysozyme, Myelin BasicProtein, Myoglobin, Neuron-Specific Enolase, Placental Lactogen,Prealbumin, Pregnancy assoc Plasma Protein A, Pregnancy specific beta 1glycoprotein (SP1), Prostate Specific Antigen PSA, PSA-A1-Act complex,Prostatic Acid Phosphatase PAP, Proteinase 3 (PR3/Anca), Prothrombin,Retinol Binding Globulin RBP, recombinant human RO/SS-A 52 kda,recombinant human RO SS-A 60 kda, Sex Hormone Binding Globulin SHBG,S100 (BB/AB), S100 BB homodimer, Thyroglobulin Tg, Thyroid MicrosomalAntigen, recombinant thyroid peroxidase TPO, Thyroid Peroxidase TPO,Thyroxine Binding Globulin TBG, Transferrin, Transferrin receptor,Troponin I complex, Troponin C, Troponin I, Troponin T, Urine Protein 1.

Plasma or serum proteins may also encompass proteins of external origin,which are not necessarily forming part of the common physiologicalpopulation, but may be found in the body, i.e. proteins from diet originor from drug compositions. Preferably, plasma or serum proteins arehuman α₁-acid glycoprotein (AAG), human serum albumin (HSA), human serum(HS), and lipoproteins, which are proteins mostly involved in thebinding of HIV antivirals in plasma or serum Human AAG is an acute-phaseprotein whose expression increases during acute inflammatory episodes,infections, injuries, neoplastic disease, and AIDS (Kremer et al,Pharmacol Rev. 1988, 40: 1-47; Oie et al, 1993, J Acquir Immune DeficSyndr Hum Retrovirol. 5:531-533; Mackiewicz et al, 1995, Glycoconj. J.12:241-247; van Dijk et al, 1995, Glycoconj J. 12:227-233). The level ofAAG in human serum fluctuates between 0.15 and 1.5 mg/mL, and theaverage value may vary by as much as 4-fold between healthy volunteersand AIDS patients (Kremer et al, Pharnacol Rev. 1988, 40: 1-47; Oie etal, 1993, J Acquir Immune Defic Syndr Hum Retrovirol. 5:531-533).Additionally, AAG concentrations have been suggested to vary by race orethnicity (Johnson et al, 1997, J. Pharm. Sci. 86: 1328-1333). It hasbeen reported that AAG exists as a mixture of two or three geneticvariants (the A variant and the F1 and/or S variants) in the plasma orserum of most individuals (Herve et al, 1998, Mol. Pharmacol.54:129-138), which present different drug binding specificities.

Human serum albumin (HSA) is quite an abundant protein in the bloodstream—it is present at a concentration of about 40 mg/ml. The primaryfunction of HSA is to act as a transporter of fatty acid molecules. HSAhas multifunctional binding properties which range from various metals,calcium and copper, to fatty acids, hormones and therapeutic drugs.

Particular plasma or serum proteins may have several variants. The termprotein variant refers to a polypeptide comprising one or moresubstitutions of the specified amino acid residues underlying theprotein. The total number of such substitutions is typically not morethan 10, e.g. one, two, three, four, five or six of said substitutions.In addition, the protein variant may optionally include othermodifications of the parent enzyme, typically not more than 10, e.g. notmore than 5 such modifications. The variant generally has a homologywith the parent enzyme of at least 80%, e.g. at least 85%, typically atleast 90% or at least 95%. Variants may not only differ in primarystructure (amino acid sequence), but also in secondary or tertiarystructure and the amount and structure of covalently attachedcarbohydrates. A protein may be present in plasma or serum in differentvariants, at similar or different concentrations. Variants of a proteinmay exhibit different binding properties. For instance human α-1 acidglycoprotein is present in two different variants, A and F1/S, whichhave different binding properties to various ligands and drugs. Humanserum albumin may be present as different mutants such as K195M, K199M,F211V, W214L, R218M, R222M, H242V, and R257M.

The methods of the present invention may additionally comprise as partof the test composition, any compound, including, but not limited to,dipeptides, tripeptides, polypeptides, proteins, small and large organicmolecules, buffers, or test aid components, and derivatives thereof.

In a particular embodiment, the method preferably includes a competitivebinding agent. A competitive binding agent refers to those moleculesthat competitively bind to plasma or serum proteins in the presence ofHIV inhibitors. Said competitive binding agent could be one or two moredrugs, preferably other drugs than antivirals which bind to plasma orserum proteins, also preferably one or two more drugs effective to treatAIDS and related syndromes, more preferably one or two more antivirals,such as NNRTI, NRTI, PI, fusion inhibitors, entry inhibitors, integraseinhibitors, as well preferably, the compound ritonavir, so concomitantadministration of antiretrovirals, optionally with other types of drugs,and its influence on plasma or serum protein binding properties may bestudied. Therefore, the present invention also provides a method foridentifying compounds that bind competitively to plasma or serumproteins in the presence of HIV inhibitors. Said compounds may be usedas co-administered agents to increase the free plasma or serumconcentration of protease inhibitors.

In an alternative embodiment, the method includes a binding enhancingagent. A binding enhancing agent refers to those molecules that enhancethe binding of antiretrovirals to plasma or serum proteins. Thus, thepresent invention provides a method for identifying compounds thatenhance the binding of HIV inhibitors to plasma or serum proteins. Saidcompounds may be used as co-administered agents to improve management oftherapeutic concentrations of HIV inhibitors.

The inhibitory activities, fold-changes and ratios thereof, exhibited bythe different HIV inhibitors in the presence of various viral strains,and in the presence and absence of plasma or serum proteins, areparticularly useful values to establish the pharmacokineticcharacteristics of the antivirals such as the distribution volume,half-life, bioavailability, and to further determine the therapeuticamount, dosage amounts and dosage intervals, necessary to accomplish aneffective therapeutic treatment. Same information may be used forpatient management, therapeutic drug monitoring, thus, to adjust andtailor the dosage regimen for individual patients and conditions. Thus,the present invention further provides a method for pharmacokineticallycharacterizing HIV inhibitors, comprising:

-   -   i) determining the inhibitory activity of at least one HIV        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one HIV        inhibitor in a cellular assay in the absence of the human plasma        or serum proteins against the at least one HIV virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and ii);    -   iv) determining the inhibitory activity of the at least one HI        inhibitor against at least one HIV virus strain of a patient;    -   v) multiplying the ratio obtained in iii) by the inhibitory        activity determined in iv); and    -   vi) using the inhibitory activity as determined in v) to        calculate physiological therapeutic dosages; and    -   wherein said at least one HIV virus strain in i) and ii) has        been selected to be inhibited by said at least one HIV inhibitor        with an inhibitory activity which falls within the range of        plasma concentrations of said at least one HIV inhibitor when        used at therapeutic dosages.

In a similar embodiment, the present invention provides a method forpharmacokinetically characterizing protease inhibitors, comprising:

-   -   i) determining the inhibitory activity of at least one protease        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one        protease inhibitor in a cellular assay in the absence of the        human plasma or serum proteins against the at least one HIV        virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and ii);    -   iv) determining the inhibitory activity of the at least one        protease inhibitor against at least one HIV virus strain of a        patient;    -   v) multiplying the ratio obtained in iii) by the inhibitory        activity determined in iv); and    -   vi) using the inhibitory activity as determined in v) to        calculate physiological therapeutic dosages; and    -   wherein said at least one HIV virus strain in i) and ii) has        been selected to be inhibited by said at least one protease        inhibitor with an inhibitory activity which falls within the        range of plasma concentrations of said at least one protease        inhibitor when used at therapeutic dosages.

The present invention further provides a method of constructing apharmacokinetic profile database of HIV inhibitors, with the influenceof plasma or serum protein binding, comprising:

-   -   i) determining the inhibitory activity of at least one HIV        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one HIV        inhibitor in a cellular assay in the absence of the human plasma        or serum proteins against the at least one HIV virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and ii);    -   iv) determining the influence of human plasma or serum protein        binding on said at least one HIV inhibitor based on the ratio        obtained in iii);    -   v) determining the inhibitory activity of the at least one HIV        inhibitor against at least one HIV virus strain of a patient;    -   vi) multiplying the ratio obtained in iii) by the inhibitory        activity determined in v),    -   vii) using the inhibitory activity as determined in v) to        calculate physiological therapeutic dosages; and    -   viii) correlating in a data table the influence of human plasma        or serum protein binding of HIV inhibitors as determined in iv)        with the physiological therapeutic dosages as determined in        vii); and    -   wherein said at least one HIV virus strain in i) and ii) has        been selected to be inhibited by said at least one HIV inhibitor        with an inhibitory activity which falls within the range of        plasma concentrations of said at least one HIV inhibitor when        used at therapeutic dosages.

Said method for constructing such database also encompasses reports thatare generated comprising a listing, analysis, or other informationregarding the influence of plasma or serum protein binding,pharmacokinetic parameters, and their correlation to drug dosageregimens.

The method of this invention has further applications in the preclinicalevaluation and selection of new antivirals for future clinicaldevelopment. As such, the present invention additionally comprises amethod for measuring the influence of plasma or serum protein binding onnew compounds, comprising:

-   -   i) determining the inhibitory activity of at least one HIV        inhibitor in a cellular assay in the presence of human plasma or        serum proteins against at least one HIV virus strain;    -   ii) determining the inhibitory activity of the at least one HIV        inhibitor in a cellular assay in the absence of the human plasma        or serum proteins against the at least one HIV virus strain;    -   iii) calculating the ratio of inhibitory activities determined        in i) and ii); and    -   iv) determining the influence of human plasma or serum protein        binding on said at least one HIV inhibitor based on the ratio        obtained in iii); and    -   wherein said at least one HIV virus strain has been selected to        be inhibited by said at least one HIV inhibitor with an        inhibitory activity which falls within the range of plasma        concentrations of said at least one HIV inhibitor when used at        therapeutic dosages.

The methods provided in the present invention may optionally be used asor comprise part of a high-throughput screening assay where numeroustest compositions are evaluated for the effect of plasma or serumprotein binding to HIV inhibitors and for the pharmacokinetic derivedproperties of said HIV inhibitors.

The order of the steps of the methods of the invention may be varied.One of skill in the art would be able to determine which variations inthe order of the steps are applicable.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the claims.

EXAMPLE 1 Influence of Human Plasma or Serum Proteins on Activity ofCurrent PIs Against Wild-Type HIV-1

The influence of AAG, Human Serum Albumin (HSA) or Human Serum (HS) onthe activity of antiretrovirals was measured in a cell-based antiviralassay. A phenotypic assay was performed to measure the ability ofresistant virus strains to grow in the presence of each drug ofinterest, and in the presence of various combinations of plasma or serumproteins at different concentrations:

-   -   50% heat-inactivated human serum (HS) with or without 10% fetal        calf serum (FCS)    -   1 mg/mL AAG and 10% FCS    -   45 mg/mL human serum albumin (HSA) and 10% FCS

Anti-HIV activity was determined in HIV-or mock-infected MT4 cells bythe MTT method as described by Pauwels et al (1988),J.Virol.Meth.20:399-321, and Hertogs et al (1998),Antimicrob.Ag.Chemother.42:269-27. Briefly, recombinant ornon-recombinant virus, with a specific resistance profile, were grown incell culture to obtain viral stocks of known concentration.Susceptibility testing of the viral stocks in the presence of variousantiviral agents and in the presence of plasma or serum proteins, and adetection system based on MTT determined to which extent agentsinhibited replication of the virus strains.

Virus strains used for these studies had different origins:

-   -   HIV strains obtained by in vitro selection in the presence of        various antivirals    -   Recombinant clinical isolates constructed according to the        Antivirogram™ method as described by Hertogs et al (1998).

With the exception of indinavir, which remained unaffected, all testedPIs showed a decrease in potency against wild-type HIV-1 in the presenceof AAG and HS, but not of HSA. The decrease ranged from 5-to 75-fold andwas proportional to the AAG concentration. Virus strains with variouslevels of resistance against each of the PIs were used in similarexperiments with AAG. The results obtained showed that the decrease inpotency observed in the presence of AAG for the tested PIs was inverselyproportional to the EC₅₀ value in the absence of the protein. Atmicromolar concentrations (1-5 μM), saquinavir, ritonavir, nelfinavir,amprenavir and compound 1 showed only a less than 5-fold decrease inpotency in the presence of 1 mg/mL AAG. So it was concluded that theinfluence of AAG decreased with increasing concentrations of drugs.TABLE 1 Influence of Human Plasma Proteins on Activity of Current PIsagainst Wild Type HIV-1 Results in table 1 are expressed as the ratiobetween the EC₅₀ value determined in the presence of the indicated humanplasma or serum protein and the EC₅₀ value determined in the presence of10% FCS, against wild type HIV-1 (LAI). Results are median of at leasttwo determinations. FCS Compound (10%) AAG 1 mg/mL HSA 45 mg/mL HS 50%INDINAVIR 1 1 2 3 SAQUINAVIR 1 5 3 5 NELFINAVIR 1 31 7 25 RITONAVIR 1 185 24 LOPINAVIR 1 26 5 5 AMPRENAVIR 1 25 2 12

TABLE 2 EC₅₀ ratios, EC₅₀values determined in the presence of humanplasma or serum protein and in the presence of 10% fetal calf serum(FCS), against various resistant strains. Results in table 2 areexpressed as the ratio between the EC₅₀ value determined in the presenceof the indicated human plasma or serum protein and the EC₅₀ valuedetermined in the presence of 10% FCS, against various HIV-1 strains.Results are median of at least two determinations. in vitro testingclinically relevant concentrations concentrations sub-therapeutic (wildtype HIV) concentration 5 μM- concentrations <5 range >5 μM 500 nM 500nM-50 nM 50 nM-5 nM nM Saquinavir 2 3 4 5 19 Ritonavir 1 4 8 7 —Indinavir — 2 1 2 — Nelfinavir — 5 9 11 — amprenavir — 5 14 38 —Lopinavir — 4 20 64 — compound 1 4 7 12 24 85

Compound 1 is a PI with the following formula:

EXAMPLE 2 Influence of AAG on the Anti-HIV Activity of Saquinavir (SQV),a Highly Protein-Bound PI

Saquinavir was tested against different HIV-1 strains with varioussusceptibility (EC₅₀ values) to the inhibitor in the indicatedconcentration range. One curve represents the data obtained in thepresence of 10% FCS. One curve represents the data calculated for thedifferent strains based on the decrease observed for SQV activityagainst wild type HIV-1 in the presence of AAG (1 mg/mL). Another curverepresents the data obtained for the different strains in the presenceof AAG (1 mg/mL).

See FIG. 1.

The influence of AAG on the anti-HIV activity of SQV decreases withincreasing inhibitor concentrations.

EXAMPLE 3 Influence of AAG on the Anti-HIV Activity of Indinavir, aLowly Protein-Bound PI

Indinavir was tested against different HIV-1 strains with varioussusceptibility (EC₅₀ values) to the inhibitor in the indicatedconcentration range. One curve represents the data obtained in thepresence of 10% FCS. One curve represents the data calculated for thedifferent strains based on the decrease observed for IDV activityagainst wild type HIV-1 in the presence of AAG (1 mg/mL). Another curverepresents the data obtained for the different strains in the presenceof AAG (1 mg/mL).

See FIG. 2.

At clinically relevant inhibitor concentrations, the influence of AAG onanti-HIV activity of PIs is minimized, thereby showing the saturation ofthe interaction of the drugs with the protein.

1. A method for determining the influence of human plasma or serumprotein binding on antiretroviral therapy, comprising: i) determiningthe inhibitory activity of at least one HIV inhibitor in a cellularassay in the presence of human plasma or serum proteins against at leastone HIV virus strain; ii) determining the inhibitory activity of the atleast one HIV inhibitor in a cellular assay in the absence of the humanplasma or serum proteins against the at least one HIV virus strain; iii)calculating the ratio of inhibitory activities determined in i) and ii);and ii) determining the influence of human plasma or serum proteinbinding on said at least one HIV inhibitor based on the ratio obtainedin iii); and wherein said at least one HIV virus strain has beenselected to be inhibited by said at least one HIV inhibitor with aninhibitory activity which falls within the range of plasmaconcentrations of said at least one HIV inhibitor when used attherapeutic dosages.
 2. A method for determining the influence of humanplasma or serum protein binding on antiretroviral therapy, comprising:i) determining the inhibitory activities of at least one proteaseinhibitor in a cellular assay in the presence of human plasma or serumproteins against at least one HIV virus strain; ii) determining theinhibitory activities of the at least one protease inhibitor in acellular assay in the absence of the human plasma or serum proteinsagainst the at least one HIV virus strain; iii) calculating the ratio ofinhibitory activities determined in i) and ii); and iii) determining theinfluence of human plasma or serum protein binding on said at least oneprotease inhibitor based on the ratio obtained in iii); and wherein saidat least one HIV virus strain has been selected to be inhibited by saidat least one protease inhibitor with an inhibitory activity which fallswithin the range of plasma concentrations of said at least one proteaseinhibitor when used at therapeutic dosages.
 3. A method according toclaim 1, wherein the plasma or serum proteins are chosen from humanserum, albumin, α₁-acid glycoprotein, lipoproteins, and variantsthereof.
 4. A method according to claim 1, wherein the method furthercomprises at least one competitive binding agent or at least one bindingenhancing agent.
 5. A method of identifying compounds that bindcompetitively to plasma or serum proteins in the presence of HIVinhibitors, said method based on determining the influence of humanplasma or serum protein binding on antiretroviral therapy according toclaim
 1. 6. A method of identifying compounds that enhance binding ofHIV inhibitors to plasma or serum proteins, said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 1. 7. A method forpharmacologically characterizing HIV inhibitors comprising: i)determining the inhibitory activity of at least one HIV inhibitor in acellular assay in the presence of human plasma or serum proteins againstat least one HIV virus strain; ii) determining the inhibitory activityof the at least one HIV inhibitor in a cellular assay in the absence ofthe human plasma or serum proteins against the at least one HIV virusstrain; iii) calculating the ratio of inhibitory activities determinedin i) and ii); iv) determining the inhibitory activity of the at leastone HIV inhibitor against at least one HIV virus strain of a patient; v)multiplying the ratio obtained in iii) by the inhibitory activitydetermined in iv); and vi) using the inhibitory activity as determinedin v) to calculate physiological therapeutic dosages; and wherein saidat least one HIV virus strain in i) and ii) has been selected to beinhibited by said at least one HIV inhibitor with an inhibitory activitywhich falls within the range of plasma concentrations of said at leastone HIV inhibitor when used at therapeutic dosages.
 8. A method forpharmacokinetically characterizing protease inhibitors comprising: i)determining the inhibitory activity of at least one protease inhibitorin a cellular assay in the presence of human plasma or serum proteinsagainst at least one HIV virus strain; ii) determining the inhibitoryactivity of the at least one protease inhibitor in a cellular assay inthe absence of the human plasma or serum proteins against the at leastone HIV virus strain; iii) calculating the ratio of inhibitoryactivities determined in i) and ii); iv) determining the inhibitoryactivity of the at least one protease inhibitor against at least one HIVvirus strain of a patient; v) multiplying the ratio obtained in iii) bythe inhibitory activity determined in iv); and vi) using the inhibitoryactivity as determined in v) to calculate physiological therapeuticdosages; and wherein said at least one HIV virus strain in i) and ii)has been selected to be inhibited by said at least one proteaseinhibitor with an inhibitory activity which falls within the range ofplasma concentrations of said at least one protease inhibitor when usedat therapeutic dosages.
 9. A method of constructing a pharmacokineticprofile database of HIV inhibitors, with the influence of plasma orserum protein binding, comprising: i) determining the inhibitoryactivity of at least one HIV inhibitor in a cellular assay in thepresence of human plasma or serum proteins against at least one HIVvirus strain; ii) determining the inhibitory activity of the at leastone HIV inhibitor in a cellular assay in the absence of the human plasmaor serum proteins against the at least one HIV virus strain; iii)calculating the ratio of inhibitory activities determined in i) and ii);iv) determining the influence of human plasma or serum protein bindingon said at least one HIV inhibitor based on the ratio obtained in iii);V) determining the inhibitory activity of the at least one HIV inhibitoragainst at least one HIV virus strain of a patient; vi) multiplying theratio obtained in iii) by the inhibitory activity determined in v); vii)using the inhibitory activity as determined in v) to calculatephysiological therapeutic dosages; and viii) correlating in a data tablethe influence of human plasma or serum protein binding of HIV inhibitorsas determined in iv) with the physiological therapeutic dosages asdetermined in vii); and wherein said at least one HIV virus strain in i)and ii) has been selected to be inhibited by said at least one HIVinhibitor with an inhibitory activity which falls within the range ofplasma concentrations of said at least one HIV inhibitor when used attherapeutic dosages.
 10. A method for measuring the influence of plasmaor serum protein binding on new compounds comprising: i) determining theinhibitory activity of at least one HIV inhibitor in a cellular assay inthe presence of human plasma or serum proteins against at least one HIVvirus strain; ii) determining the inhibitory activity of the at leastone HIV inhibitor in a cellular assay in the absence of the human plasmaor serum proteins against the at least one HIV virus strain; iii)calculating the ratio of inhibitory activities determined in i) and ii);and iv) determining the influence of human plasma or serum proteinbinding on said at least one HIV inhibitor based on the ratio obtainedin iii); and wherein said at least one HIV virus strain in has beenselected to be inhibited by said at least one HIV inhibitor with aninhibitory activity which falls within the range of plasmaconcentrations of said at least one HIV inhibitor when used attherapeutic dosages.
 11. A method according to claim 1 suitable for highthroughput screening.
 12. The method according to claim 2, wherein theplasma or serum proteins are chosen from human serum, albumin, α1-acidglycoprotein, lipoproteins, and variants thereof.
 13. The methodaccording to claim 2, wherein the method further comprises at least onecompetitive binding agent or at least one binding enhancing agent. 14.The method according to claim 3, wherein the method further comprises atleast one competitive binding agent or at least one binding enhancingagent.
 15. The method of identifying compounds that bind competitivelyto plasma or serum proteins in the presence of HIV inhibitors, saidmethod based on determining the influence of human plasma or serumprotein binding on antiretroviral therapy according to claim
 2. 16. Themethod of identifying compounds that bind competitively to plasma orserum proteins in the presence of HIV inhibitors, said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 3. 17. The method ofidentifying compounds that bind competitively to plasma or serumproteins in the presence of HIV inhibitors, said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 4. 18. The method ofidentifying compounds that bind competitively to plasma or serumproteins in the presence of HIV inhibitors, said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 12. 19. The method ofidentifying compounds that bind competitively to plasma or serumproteins in the presence of HIV inhibitors, said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 13. 20. The method ofidentifying compounds that bind competitively to plasma or serumproteins in the presence of HIV inhibitors said method based ondetermining the influence of human plasma or serum protein binding onantiretroviral therapy according to claim
 14. 21. The method accordingto claim 2 suitable for high throughput screening.
 22. The methodaccording to claim 3 suitable for high throughput screening.
 23. Themethod according to claim 4 suitable for high throughput screening. 24.The method according to claim 5 suitable for high throughput screening.25. The method according to claim 6 suitable for high throughputscreening.
 26. The method according to claim 7 suitable for highthroughput screening.
 27. The method according to claim 8 suitable forhigh throughput screening.
 28. The method according to claim 9 suitablefor high throughput screening.
 29. The method according to claim 10suitable for high throughput screening.
 30. The method according toclaim 11 suitable for high throughput screening.
 31. The methodaccording to claim 12 suitable for high throughput screening.
 32. Themethod according to claim 13 suitable for high throughput screening. 33.The method according to claim 14 suitable for high throughput screening.34. The method according to claim 15 suitable for high throughputscreening.
 35. The method according to claim 16 suitable for highthroughput screening.
 36. The method according to claim 17 suitable forhigh throughput screening.
 37. The method according to claim 18 suitablefor high throughput screening.
 38. The method according to claim 19suitable for high throughput screening.
 39. The method according toclaim 20 suitable for high throughput screening.